Compositions and methods for promoting myocardial and peripheral angiogenesis

ABSTRACT

Methods, compositions and devices are disclosed for use in growing new blood vessels to restore or improve blood flow to ischemic tissues and organs of the body. Compositions comprising IGD peptides, particularly GGIGDGG, are able to induce migration in human endothelial cells and promote vessel formation in an in vitro model assay system.

This application is a divisional of U.S. application Ser. No.10/027,015, filed Dec. 21, 2001, now U.S. Pat. No. 7,232,802.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to the treatment of ischemicconditions of major organs in the human body by promoting growth ofcollateral vessels to increase blood flow to the target organs andtissues. More specifically, the invention relates to peptide basedangiogenic compositions, methods and devices for treating cardiovasculardisease associated with reduced blood flow arising from narrowing of anative blood vessel or occlusion of a bypass graft.

2. Description of Related Art

In the United States, cardiac failure due to underlying coronary heartdisease is currently one of the leading causes of death. At the presenttime, coronary artery bypass graft (CABG) surgery and percutaneoustransluminal coronary angioplasty (PTCA) are the most widely usedinterventions for treating advanced cardiac disease. In CABG, anautologous vessel is used to bypass the area of coronary obstruction orocclusion and to restore the blood flow. In PTCA, a catheter device isemployed to unblock the clogged blood vessel to restore adequate bloodflow to the heart and a metal stent is usually implanted to maintainvessel patency. Both of those procedures are considered to be highlyinvasive, are associated with a certain incidence of restenosis, and maynot be appropriate for every patient in need of relief from coronaryvessel obstructions—particularly when the patient is elderly or hasundergone a previous CABG or PTCA procedure. Moreover, in peripheralvascular disease, when the vessels that supply blood to the legs,intestines and other areas of the body experience atheroscleroticnarrowing, neither procedure may be an option because of the small sizeof the occluded peripheral vessels.

In some individuals, blood vessel occlusion is partially compensated bythe spontaneous process of angiogenesis, or new vessel growth, in whichnew collateral vessels form over a period of time to provide naturalbypasses around the occluded vessels. The process of angiogenesisgenerally involves basement membrane degradation and endothelial cellmigration and proliferation to form capillaries which may developfurther into mature vessels. Naturally occurring mitogenic factorsreleased from lymphoid and endothelial cells can induce angiogenesis andpromote neovascularization of damaged or blood starved tissue. The newlyformed vessels can oftentimes supplement or entirely replace thefunction of the impaired vessels, thereby restoring blood flow to thedeprived tissue served by the occluded vessels.

Some individuals are unable to generate sufficient collateral vessels toadequately compensate for diminished blood flow to the ischemic tissue.Therefore, a third treatment approach, still in development, endeavorsto induce or enhance the growth of new blood vessels around an area ofobstruction to restore adequate blood flow to the heart or other blooddeprived tissue. Induced or promoted angiogenesis is believed by manyinvestigators to offer the least invasive way to treat coronary heartdisease, to be suitable for use in a large percentage of the patientpopulation (including in particular some patients who are not candidatesfor either CABG or PTCA), and applicable for neovascularization of bothmyocardial and peripheral tissues.

Several angiogenic agents have recently been identified that promoteangiogenesis through either direct attraction and/or induction ofproliferation of endothelial cells, or indirect action by stimulatingother cell types (e.g., mast cells or macrophages) that, in turn,produce angiogenic factors. Examples of these agents include vascularendothelial growth factor (VEGF), osteonectin or SPARC, basic fibroblastgrowth factor (bFGF), angiogenin, endothelial growth factor (EGF),platelet derived growth factor (PDGF), transforming growth factor-alpha(TGF-α), transforming growth factor-beta (TGF-β), and tumor necrosisfactor-alpha (TNF-α). Each of these angiogenic agents or factors areeither synthetic, meaning that they are manufactured chemically fromnon-living sources, or are produced by recombinant manufacturingprocesses (Freedman, S. B., and Isner, J. M., Therapeutic angiogenesisfor ischemic cardiovascular disease, J. Mol. Cell Cardiol 33(3): 379-393(2001)).

Another angiogenic agent, disclosed in co-assigned U.S. Pat. No.6,211,157 (Benedict et al./Sulzer Biologics, Inc.), is a bone-derivedangiogenic protein (BDAP) mixture that provides a more robust angiogenicresponse than many single factors such as bFGF or VEGF.

Many approaches to enhancement of localized angiogenesis and/or woundhealing involve introduction of an extracellular matrix-like materialthat can serve as a support or scaffold at the desired site and withwhich the target cells may interact, usually via specific cell surfacereceptors, to promote cell proliferation. Extracellular matrix (“ECM”)is the structurally stable material beneath the epithelia surroundingthe cells of the connective tissue and constitutes a sort of naturalscaffolding material. ECM can also be defined as the macromolecularcomponents of connective tissue, generally consisting of proteoglycans,polysaccharides and proteins, which have major roles in cell shape, cellmigration and differentiation, and control of cell growth. A subset ofthe ECM family of proteins is the adhesion proteins. The two majoradhesion proteins, fibronectin and laminin, are involved in manycellular processes, including tissue repair, embryogenesis, bloodclotting, and cell migration/adhesion. Accordingly, various studiesdirected at providing a favorable cellular environment to promote cellproliferation involve fibronectin or particular fibronectin peptides.Many of those studies employ the Arg-Gly-Asp or RGD sequence, which ispart of the cell binding domain of fibronectin (see, e.g., U.S. Pat. No.5,677,276 (Dickerson et al.), and S. L. Schor et al., J Cell Sci109:2581-2590 (1996)).

Recently, it has been reported that the isoleucine-glycine-aspartic acid(Ile-Gly-Asp or IGD) tripeptide sequence, a component of the fibronectintype I module, can induce cell migration of dermal fibroblasts (S. L.Schor et al., J. Cell Sci 112:3879-3888 (1999)). Biological activity hasnot previously been ascribed to the conserved IGD motif in fibronectin,although previous studies have implicated the ninth type I repeat, whichcontains the IGDS (SEQ ID NO:5) sequence, in the assembly of anextracellular fibronectin matrix (M A Chernousov et al., J Biol Chem266:10851-10858 (1991)). In PCT Published Application No. WO 99/02674(Schor et al./University of Dundee), certain IGD-containing peptideswere described and the IGDS peptide was shown to increase fibroblastmigration and vessel number under certain conditions in a rat woundhealing model.

While significant advancements have been made in identifying andunderstanding various modulators of cellular migration and angiogenesis,there remains a pressing need for effective means to promoteangiogenesis at ischemic sites in the body, such as the heart andtissues fed by the peripheral vascular system, to restore circulation toblood deprived organs and tissues affected by atherosclerotic disease.

SUMMARY OF THE INVENTION

The present invention seeks to provides compositions, devices andmethods for use in growing new blood vessels to restore or improve bloodflow to ischemic tissues of the body, especially for treating cardiacand peripheral blood vessel disease.

In one embodiment of the present invention, compositions are providedfor promoting angiogenesis in a region of the body for whichangiogenesis is desired, such as an area in need of angiogenesis (e.g.,an ischemic region). In a particular embodiment, a composition isprovided comprising a protein having angiogenic activity and comprisinga domain having the amino acid sequence isoleucine-glycine-aspartic acid(“IGD” in standard amino acid letter designation). In preferredembodiments, the complete amino acid sequence of the protein comprisesfifty (50) or few amino acid residues, more preferably twenty-five (25)or fewer, more preferably still ten (10) or fewer.

In another embodiment, a composition is provided comprising a proteinhaving angiogenic activity and the amino acid sequence ofisoleucine-glycine-aspartic acid [SEQ ID NO. 1]. In yet anotherembodiment, a composition comprising a protein having angiogenicactivity and the amino acid sequenceglycine-glycine-isoleucine-glycine-aspartic acid-glycine-glycine [SEQ IDNO. 2] (“GGIDGGG”) is provided. In a further embodiment, a compositioncomprising a protein having angiogenic activity and the amino acidsequence isoleucine-glycine-aspartic acid-isoleucine-glycine-asparticacid [SEQ ID NO. 3] (“IGDIGD”) is provided. In a still furtherembodiment, a composition comprising a cyclic protein having angiogenicactivity and the amino acid sequence isoleucine-glycine-asparticacid-isoleucine-glycine-aspartic acid [SEQ ID NO. 4] (“cyclic IGDIGD”).In a yet further embodiment, a composition is provided comprising aprotein having angiogenic activity and an amino acid sequence designatedby the formula:ZZIGDZZ  (FORMULA 1)wherein I represents isoleucine, G represents glycine, D representsaspartic acid, and Z represents any of the twenty biological aminoacids. The peptides of SEQ. ID NOS. 1-4, and other representative IGDpeptides have been surprisingly found to exhibit excellent biologicalactivity, as assessed by their ability to induce migration in a varietyof cell types, including human endothelial cells, human fibroblast cellsand sheep nucleus cells.

In another embodiment, a composition comprising a protein havingangiogenic activity and the amino acid sequenceisoleucine-glycine-aspartic acid-serine [SEQ ID NO. 5] (“IGDS”) isprovided. In another embodiment, a composition comprising a proteinhaving angiogenic activity and the amino acid sequenceisoleucine-glycine-aspartic acid-glutamine [SEQ ID NO. 6] (“IGDQ”).

In another embodiment of the present invention there is provided anangiogenic composition (i.e., a composition that exhibits angiogenicactivity) comprising at least one of the peptides of SEQ ID NOS. 1-6 andFormula 1, and at least one angiogenic growth factor other than theforegoing peptide(s). In certain embodiments the other angiogenic growthfactor is a bone-derived angiogenic protein mixture (BDAP), one or morebone morphogenetic proteins (BMPs), vascular endothelial cell growthfactor (VEGF), basic fibroblast growth factor (bFGF), angiogenin,endothelial growth factor (EGF), platelet derived growth factor (PDGF),transforming growth factor-alpha (TGF-α), transforming growthfactor-beta (TGF-β), or a tumor necrosis factor-alpha (TNF-α).

According to still another embodiment of the present invention, anangiogenic composition is provided that comprises at least one of thepeptides if SEQ ID NOS. 1-6 and Formula 1, and at least one recombinantangiogenic growth factor.

In a further embodiment of the present invention, a composition isprovided that is active for promoting cell migration and/or angiogenesisunder cell growth promoting conditions. The composition comprises atleast one of the peptides of SEQ ID NOS 1-6 and Formula 1, and a matrixmaterial. In some embodiments, the composition also includes apharmacologically acceptable carrier, and the composition may besterilized for use in the body.

In certain other embodiments of the present invention, a method ofpromoting myocardial angiogenesis is provided. The method includesadministering intramyocardially to an ischemic area of the heart of anindividual in need of such treatment, a composition comprising at leastone of the peptides of SEQ ID NOS. 1-6 and Formula 1 in aphysiologically acceptable carrier, in an amount effective to enhancevascular endothelial cell migration and/or proliferation in the ischemicarea.

In another embodiment of the method, the composition that isadministered to the patient comprises, in addition to at least one ofthe above-identified peptides of SEQ ID NOS. 1-6 and Formula 1, aphysiologically acceptable carrier, and at least one of the followinggrowth factors: BDAP, one or more BMPs, VEGF, bFGF, angiogenin, EGF,PDGF, TGF-α, TGF-β, and TNF-α. In some embodiments the carrier comprisespolyvinylpyrrolidinone. In a preferred embodiment, the method includesdelivering the composition to the ischemic area by injection.

In another embodiment of the present invention, a method of promotingperipheral angiogenesis is provided, that comprises administering to anischemic area of an organ or tissue fed by a peripheral vessel of anindividual in need of treatment, a composition comprising at least oneof the peptides of SEQ ID NOS. 1-6 and Formula 1 in a physiologicallyacceptable carrier, in an amount effective to enhance vascularendothelial cell migration and/or proliferation at the ischemic area.The composition may also comprise one or more of the following growthfactors: BDAP, one or more BMPs, VEGF, bFGF, angiogenin, EGF, PDGF,TGF-α, TGF-β, and TNF-α. The physiologically acceptable carrier maycomprise polyvinylpyrrolidinone, and the method may compriseadministering the composition to the ischemic area by hypodermicinjection.

In still another embodiment of the present invention, a method ofenhancing blood flow to an ischemic tissue of the body in an individualin need of treatment is provided. The method comprises administering anangiogenic composition containing at least one of the peptides of SEQ IDNOS. 1-6 and Formula 1 in a physiologically acceptable carrier to adefined area of the ischemic tissue, in an amount effective to stimulatevascular endothelial cell migration and/or proliferation sufficient torestore or increase blood flow to the ischemic tissue. In someembodiments, the method comprises delivering the composition to a siteadjacent a native blood vessel narrowed due to atherosclerotic disease.The method may comprise delivering the composition to a site adjacent abypass graft. Some embodiments of the method comprise delivering anangiogenic composition that contains, in addition to at least one of thepeptides of SEQ ID NOS. 1-6 and Formula 1 and a carrier, at least one ofthe above-identified growth factors. These and other embodiments,features and advantages of the present invention will become apparentwith reference to the following description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate tabulated results of a cell migration assayfor selected IGD-motif tripeptides according to the protocol ofExperiment 1. FIG. 1A shows migration of human aortic endothelial cellsin response to IGD tripeptide or capped IGD tripeptide. A dose dependenteffect was observed with the optimal concentration being 100 μm for theuncapped peptide. However, with the capped peptide, better migrationcompared to the uncapped peptide was observed at a 10-fold lowerconcentration. The respective scramble peptides showed fewer migrationcells (data not shown).

FIG. 1C provides photographs showing the results of cell migrationassays for IGD-motif tripeptides as tested in Experiment 1. It shows themigration of endothelial cells in a modified Boyden chamber assay inresponse to various treatments. Both IGD and GGIGDGG cause increasedmigratory response compared to either the negative control or thecorresponding scramble peptides.

FIG. 2 shows tabulated results of a cell proliferation assay for fourIGD-motif tripeptides according to the protocol of Experiment 2. Thefigure shows proliferation of human aortic endothelial cells in responseto treatment with IGD tripeptide, the capped IGD tripeptide, and cappedand uncapped scramble tripeptide, at two different concentrations. Itcan be seen that with the capped tripeptide the proliferation responsewas nearly twice that of the uncapped tripeptide. In either case, thescramble peptides showed a significantly lower response than the activepeptides.

FIG. 3A shows tabulated results of a quail CAM assay for four IGD-motiftripeptides according to Experiment 3. The quail CAM assay showed anincrease in blood vessels in response to the peptide and the cappedpeptide compared to the respective scramble peptides. Data presentedhere is as a percent change over negative control, the negative controlbeing set at 100%. It can be seen that the capped peptide was betterthan the uncapped peptide at equivalent concentrations.

FIG. 3B provides photographs of quail CAM assay results for selectedIGD-motif tripeptides tested in Experiment 3.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In the present disclosure, the terms “isoleucine-glycine-aspartic acidpeptide,” “Ile-Gly-Asp peptide,” and “IGD containing peptide” all referto a peptide having at least one isoleucine-glycine-aspartic acidsequence, and which has cell attachment promoting activity. “IGD” is theconventional amino acid code letter designation of that sequence. In itsbroadest sense, references to an IGD-motif peptide also include peptidesthat contain isoleucine-glycine-aspartic acid peptide or demonstratecell attachment promoting properties similar to those of theisoleucine-glycine-aspartic acid sequence. Examples are Ile-Gly-Asp(IGD) [SEQ ID NO. 1]; Gly-Gly-Ile-Gly-Asp-Gly-Gly (GGIGDGG) [SEQ ID NO.2]; Ile-Gly-Asp-Ile-Gly-Asp (IGDIGD) [SEQ ID NO. 3]; cyclicIle-Gly-Asp-Ile-Gly-Asp (cyclic IGDIGD) [SEQ ID NO. 4]; Ile-Gly-Asp-Ser(IGDS) [SEQ ID NO. 5]; and Ile-Gly-Asp-Gln (IGDQ) [SEQ ID NO. 6]. Thestructures of these peptides are shown in the attached sequence listing.Other examples include peptides having an amino acid sequencecharacterized by Formula 1, or peptides of 50 or few amino acid residuesand having a domain comprising the IGD sequence.

EXAMPLES

The peptides of SEQ ID NOS. 1-6 were synthetically made using a peptidesynthesizer. Polyvinylpyrrolidinone (povidone) was obtained from IISPChemicals (Wayne, N.J.). Human endothelial cells and human fibroblastcells were obtained from Clonetics (Walkersville, Md.).

Preparation of Sterile Formulations.

Sterile formulations of the IGD peptides as specified in SEQ ID NOS. 1-6may be prepared by synthesizing the peptides in a peptide synthesizerand filter sterilizing the resulting solutions using a 0.22 micronfilter. The peptides can be lyophilized following the filtersterilization and reconstituted in 1% aqueous polyvinylpyrrolidinone orother povidone compounds as described in co-pending U.S. patentapplication Ser. No. 10/027,669, filed Dec. 21, 2001, entitled“POVIDONE-CONTAINING CARRIERS FOR POLYPEPTIDE GROWTH FACTORS,” which ishereby incorporated herein in its entirety. Alternatively, other knowncarriers such as dilute HCl (10 mM) may be used. Povidone is preferredas a carrier in conducting in vitro assays because it is not cytotoxic.

Example 1 In vitro Cell Migration Assays.

The ability of IGD-motif peptides according to the present invention topromote cell migration of human endothelial and smooth muscle cells,which are processes characteristic of angiogenesis, was evaluated byconducting cell migration assays for the peptides of SEQ ID NOS 1 and 2,and a scramble of the IGD peptide characterized by the DIG sequence. Acapped version of the scramble characterized by the GGDIGGG (SEQ IDNO:7) sequence was also tested.

Chemotaxis trays (Chemo Tx disposable migration chamber, 6 mm diameter,300 μl/well, 96 wells with 8 μm filter membranes from Neuro Probe, Inc.,Gaithersville, Md.) for evaluating cell migration of the peptides weresterilized by placing the trays under UV light overnight. Furtheroperations with the membrane were carried out under aseptic conditions.The filter membranes were loaded with gelatin to provide a suitableenvironment for the cells testing by soaking in 3% acetic acid overnightand then for 2 hours in 0.1 mg/ml gelatin. They were then rinsed insterile water and allowed to air dry. Such membranes may be stored atroom temperature for up to 1 month.

The cells to be used in the assay (endothelial cells or smooth musclecells) were starved for 24 hours before use in appropriate culture mediacontaining 0.1% Fetal Bovine Serum (“FBS”) instead of the customary 10%FBS serum, and 1× penicillin-streptomycin antibiotics. The wells of the96 well chamber of the chemotaxis unit were filled with media containing0.1% serum alone or 0.1% serum and the test material (control orchemoattractant respectively). The filter membrane was positioned overthe plate, aligning the holes in the corners of the frame with the fourpins on the microplate, and the membrane was snapped into place makingsure that the media in the wells touched the membrane completely. Fifty(50) μl of cell suspension in the starvation-media, at a concentrationof 4×10⁴ viable cells per charge were plated onto each site (over eachwell). The plate was incubated at 37° C. in an atmosphere of 5% CO₂ for4 hours.

After incubation the lid was removed and with the filter still in placethe cells on the upper surface of the membrane were gently wiped off andwashed by carefully flushing the top surface of the filter with media byholding the plate with filter at a 45° angle over a container. The cellson the undersurface of the membrane were then fixed in methanol (˜20minutes) and stain with Diff-Quik Staining Set. The membrane was thenallowed to dry and the number of cells that migrated through the filterpores was determined by counting the number in a field under a lightmicroscope.

The results indicate that both the peptide of SEQ ID NO. 1 and SEQ IDNO. 2 promoted cell migration, for both the human aortic endothelialcells and the human smooth muscle cells. Neither the DIG scramble northe capped GGDIGGG scramble (SEQ ID NO:7) showed cell migrationsignificantly greater than controls. See FIGS. 1A, 1B and 1C.

Example 2 In Vitro Cell Proliferation Assays.

The capacity of IGD-motif peptides according to the present invention topromote cell migration of human aortic endothelial cells was evaluatedby conducting in vitro cell proliferation assays for the peptide of SEQID NOS 1 and 2, the DIG scramble peptide, and the capped version of thescramble, GGDIGGG (SEQ ID NO:7). Human aortic endothelial cells grown to˜95% confluency were seeded (5000 cells/well) in growth medium for fourhours to allow cells to adhere. The cells were then transferred to thestarvation medium described in Example 1 and starved for about 18 hours.The cells were then transferred to starvation medium containing the testpeptide, and the cells were allowed to proliferate for an additional 48hours. The medium was then removed and the wells were washed with PBS.The cells were then subjected to a single freeze-thaw cycle. CyQuantreagent (Molecular Probes, Eugene, Oreg.) was then added to the cellsaccording to the manufacturer's instructions and the cells incubated for5 minutes in the dark. The intensity of the color—which is directlyproportional to the number of cells—is read at an excitation wavelengthof 485 nm and the emission wavelength set at 535 nm.

The results indicate that both the peptide of SEQ ID NO. 1 and SEQ IDNO. 2 promoted cell proliferation of human aortic endothelial cells.Neither the DIG scramble nor the capped GGDIGGG scramble (SEQ ID NO:7),in contrast, showed cell migration significantly greater than controls.See FIG. 2.

Example 3 Quail Chorioallantoic Membrane (CAM) Angiogenesis Assay.

The activity of IGD-motif peptides for inducing migration andproliferation in endothelial or smooth muscle cells in an in vitro quailchorioallantoic membrane (CAM) model was assayed in a similar manner tothat described by Parsons-Wingerter et al., Microvascular Research55:201-214 (1998), the disclosure of which is hereby incorporated hereinby reference. Briefly, fertilized Japanese quail eggs (cotumix cotumixjaponica) were opened onto petri dishes on day 3 post-incubation. After7 days of culture, the four IGD-motif peptides tested in Examples 1 and2 (IGD, GGIGD (residues 1-5 of SEQ ID NO:2), DIG, GGDIGGG (SEQ ID NO:7)) each dissolved in 1% polyvinyl pyrrolidine prewarmed to 37° C., weredistributed evenly onto the surface of a CAM in separate petri dishes.After 24 hours of incubation, the CAM's were fixed, dissected andphotographed at 10× magnification to visualize the arterial vasculartree, including endstage vessels. Digital images of triplicate CAMspecimens were acquired at 10× magnification in grayscale, binarized toblack-and-white, and skeletonized. The vessel branching pattern wasanalyzed and quantified by the fractal dimension.

The results indicate that both the IGD and GGIGDGG (SEQ ID NO:2)peptides promoted angiogenesis in the CAM model, while neither the DIGscramble nor the GGDIGGG (SEQ ID NO:7) capped scramble promotedangiogenesis significantly better than controls. See FIGS. 3A and 3B.

The foregoing in vitro data strongly suggest that IGD-motif peptideswill be angiogenic in known animal models involving, e.g., dogs orrabbits as well as in similar human clinical situations. An increase inblood vessel density, size and maturity of the vessels can beanticipated as outcomes of the studies in the animal models. Thespecificity to the peptide is clear, in view of the inability of thescrambled peptide to provoke a similar positive response in the in vitrostudies.

In some instances, where either coronary or peripheral angiogenesis isdesired, it may be preferable to also include a cell growth promotingmatrix material with the IGD peptide injection composition in order tofurther enhance cell migration or recruitment and proliferation.Suitable matrix materials include polyvinylpyrrolidinone and diluteacidic solutions, e.g. 10 mmol HCl. The IGD peptide-matrix mixture ispreferably introduced at the ischemic site where vascularization isdesired.

Another alternative angiogenesis promoting compositions may comprise anIGD-motif peptide, such as any of the peptides of SEQ ID NOS. 1-6 orFormula 1, combined with a known angiogenic substance such as BDAP, oneor more BMPs, VEGF, bFGF, angiogenin, EGF, PDGF, TGF-α, TGF-β, andTNF-αA preferred angiogenic composition comprises an IGD-motif peptidesuch as GGIGDGG and the bone-derived angiogenic protein mixture (BDAP)described in co-assigned U.S. Pat. No. 6,211,157. In another preferredembodiment, the composition comprises a mixture of at least oneIGD-motif peptide of SEQ ID NOS. 1-6 and Formula 1 and VEGF. Theresulting vascular endothelial cell migration stimulating effect and/orcell proliferation effect of the combination is expected to be additiveor even synergistic compared to the effects of either the IGD peptide orthe other growth factor alone. Either during embryologic development orduring tissue regeneration in vivo, in non fetal tissues, several growthfactors are upregulated—some simultaneously and otherssequentially—indicating the involvement of more than one factor for thecompletion of the process. Some of the earlier clinical studiesaddressing the ability of single growth factors to induce angiogenesishave not been completely successful, further emphasizing the need formore than a single factor or signal transduction pathway.

In addition to the representative IGD-motif peptides discussed in thepreceding examples, one could also or instead, under suitablecircumstances, employ another cell migration stimulating IGD-motifpeptide in the treatment methods described herein. Such IGD peptides aredescribed in PCT Published Application No. WO 99/02674, which is herebyincorporated herein by reference.

While the preferred embodiments of the invention have been shown anddescribed, modifications thereof can be made by one skilled in the artwithout departing from the spirit and teachings of the invention. Theembodiments described herein are exemplary only, and are not intended tobe limiting. Many variations and modifications of the inventiondisclosed herein are possible and are within the scope of the invention.Accordingly, the scope of protection is not limited by the descriptionor examples set out above, but is only limited by the claims whichfollow, that scope including all equivalents of the subject matter ofthe claims. Each and every claim is incorporated into the specificationas an embodiment of the present invention. Thus the claims are a furtherdescription and are an addition to the preferred embodiments of thepresent invention.

1. A method of enhancing blood flow to a tissue of a human body, comprising administering an effective amount of an angiogenic composition containing at least one peptide comprising GGIGDGG (SEQ ID NO:2) in a physiologically acceptable carrier to a defined area of the tissue in the human body in need of such treatment, wherein the at least one peptide stimulates or induces migration, attachment, and/or proliferation of vascular endothelial or smooth muscle cells sufficient to increase blood flow to the tissue.
 2. The method of claim 1 wherein the administering comprises delivering said composition to a site adjacent a native blood vessel narrowed due to atherosclerotic disease.
 3. The method of claim 1 wherein the administering comprises delivering the composition to a site adjacent a bypass graft.
 4. The method of claim 1, wherein the angiogenic composition comprises at least one angiogenic growth factor other than the at least one peptide.
 5. The method of claim 4, wherein the other at least one angiogenic growth factor is chosen from the group consisting of bone-derived angiogenic proteins (BDAPs), vascular endothelial cell growth factor (VEGF), basic fibroblast growth factor (bFGF), angiogenin, endothelial growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factor-alpha (TGF-α), transforming growth factor-beta (TGF-β), and tumor necrosis factor-alpha (TNF-α).
 6. The method of claim 5, wherein the other at least one angiogenic growth factor is recombinant.
 7. The method of claim 1, wherein the angiogenic composition comprises a matrix material.
 8. The method of claim 1, wherein the tissue is ischemic.
 9. The method of claim 1, wherein administering comprises delivering the composition intramyocardially.
 10. The method of claim 1, wherein administering comprises delivering the composition to a tissue fed by peripheral vessel. 